Beta-amyloid peptide production inhibitors and methods for their identification

Abstract

A method for identifying compounds capable of inhibiting the production of β-amyloid peptide in cells comprises exposing cultured cells in one or more test compounds. The cells are cultured under conditions which produce amyloid precursor protein and which result in intracellular accumulation of an approximately 22 kD polypeptide which includes the entire sequence of the β-amyloid peptide. Test compounds which cause a change in the accumulation of the 22 kD polypeptide are considered likely candidates for use as drugs for treating β-amyloid diseases, such as Alzheimer's disease.

What is claimed is: 
     
       1. A method for identifying β-amyloid production inhibitors, said method comprising: culturing mammalian cells which produce amyloid precursor protein in the presence of a protease inhibitor present in an amount sufficient to cause intracellular accumulation of an approximately 22 kD preamyloid intermediate which can be recognized by an antibody that recognizes a carboxy-terminal fragment of amyloid precursor protein containing the entire β-amyloid peptide sequence;   introducing to the cultured cells a plurality of test compounds, wherein each test compound is introduced to a separate population of said cultured cells; and   identifying test compounds which cause a change in the accumulation of the approximately 22 kD preamyloid intermediate,   whereby test compounds which cause a change are candidates as β-amyloid production inhibitors.   
     
     
       2. A method as in claim 1, wherein the cells are from a human cell line. 
     
     
       3. A method as in claim 2, wherein the cells have been modified to overexpress amyloid precursor protein. 
     
     
       4. A method as in claim 1, wherein the cells are cultured in the presence of a protease inhibitor selected from the group consisting of leupeptin, E-64, and L-transepoxysuccinyl-L-leucylamido-3-methyl-butaine present in an amount sufficient to cause the intracellular accumulation of the approximately 22 kD preamyloid intermediate. 
     
     
       5. A method as in claim 4 wherein the protease inhibitor comprises both a cysteine protease inhibitor and an aspartic protease inhibitor. 
     
     
       6. A method as in claim 4, wherein the protease inhibitor is present at a concentration in the range from 0.1 μM to 1 mM. 
     
     
       7. A method as in claim 1, wherein the cells are exposed to the test compounds at concentrations from 1 μM to 1 mM. 
     
     
       8. A method as in claim 4, wherein the cells are exposed concurrently to the protease inhibitor and each test compound. 
     
     
       9. A method as in claim 1 wherein the test compounds comprise biological polymers selected from the group consisting of polypeptides, polysaccharides, and polynucleotides. 
     
     
       10. A method as in claim 1, wherein the test compounds which cause a change in the accumulation of the approximately 22 kD preamyloid intermediate are identified by comparing the accumulation of the approximately 22 kD preamyloid intermediate in a population of the cells exposed to a test compound to the accumulation in a population of the cells free from the test compound. 
     
     
       11. A method as in claim 10, wherein the change is increased accumulation in cells exposed to the test compound compared to accumulation in the absence of the test compound. 
     
     
       12. A method as in claim 10, wherein the change is decreased accumulation in cells exposed to the test compound compared to accumulation in the absence of the test compound. 
     
     
       13. A method for identifying β-amyloid production inhibitors, said method comprising: culturing mammalian cells which produce amyloid precursor protein in a growth medium;   introducing a protease inhibitor to the growth medium which enhances intracellular accumulation of an approximately 22 kD preamyloid intermediate which can be recognized by an antibody that recognizes a carboxy-terminal fragment of amyloid precursor protein containing the entire β-amyloid peptide sequence;   introducing a test compound to the growth medium; and   detecting changes in the intracellular accumulation of the approximately 22 kD preamyloid intermediate which result from the exposure of the cells to the test compound in the medium,   whereby test compounds which cause a change are candidates as β-amyloid production inhibitors.   
     
     
       14. A method as in claim 13, wherein the cells are from a human cell line. 
     
     
       15. A method as in claim 14, wherein the cells have been modified to overexpress amyloid precursor protein. 
     
     
       16. A method as in claim 13, wherein the substance comprises a protease inhibitor selected from the group consisting of leupeptin, E-64, and L-trans epoxysuccinyl-L-leucylamido-3-methyl-butiane present in an amount sufficient to cause the intracellular accumulation of the approximately 22 kD preamyloid intermediate. 
     
     
       17. A method as in claim 16, wherein the protease inhibitor comprises both a cysteine protease inhibitor and an aspartic protease inhibitor. 
     
     
       18. A method as in claim 16, wherein the protease inhibitor is present at a concentration in the range from 0.1 μM to 1 mM. 
     
     
       19. A method as in claim 13, wherein the cells are exposed to the test compound at a concentration from 1 μM to 1 mM. 
     
     
       20. A method as in claim 13, wherein the cells are exposed concurrently to the substance and the test compound. 
     
     
       21. A method as in claim 13, wherein the test compound comprises a biological polymer, selected from the group consisting of polypeptides, polynucleotides, and polysaccharides. 
     
     
       22. A method as in claim 13, wherein changes in the intracellular accumulation are detected by measuring the accumulation of the approximately 22 kD preamyloid intermediate in cultured cells exposed to the test compound, measuring the accumulation in genetically identical cells cultured under the same conditions but not exposed to the test compound, and comparing the two measured values. 
     
     
       23. A method as in claim 22, wherein exposure to the test compound results in an increase in the measured value of accumulation in the cells exposed to the test compound compared to the cells not exposed to the test compound. 
     
     
       24. A method as in claim 22, wherein exposure to the test compound results in a decrease in the measured value of accumulation in the cells exposed to the test compound compared to the cells not exposed to the test compound. 
     
     
       25. A method for assaying a test compound for the ability to inhibit β-amyloid production in cells, said method comprising: exposing the test compound to mammalian cells cultured in the presence of a protease inhibitor under conditions which cause the intracellular accumulation of an approximately 22 kD preamyloid intermediate which can be recognized by an antibody that recognizes a carboxy-terminal fragment of amyloid precursor protein containing the entire β-amyloid peptide sequence; and   detecting a change in the accumulation of the approximately 22 kD preamyloid intermediate relative to the accumulation in control cells which are cultured under the same condition but not exposed to the test compound,   whereby test compounds which cause a change are candidates as β-amyloid production inhibitors.   
     
     
       26. A method as in claim 25, wherein the cells are from a human cell line. 
     
     
       27. A method as in claim 26, wherein the cells have been modified to overexpress amyloid precursor protein. 
     
     
       28. A method as in claim 25, wherein the cells are cultured in the presence of a protease inhibitor selected from the group consisting of leupeptin, E-64, and L-transepoxysuccinyl-L-leucylamido-3-methyl-butaine present in an amount to cause the intracellular accumulation of the approximately 22 kD preamyloid intermediate. 
     
     
       29. A method as in claim 28, wherein the protease inhibitor comprises both a cysteine protease inhibitor and an aspartic protease inhibitor. 
     
     
       30. A method as in claim 28, wherein the protease inhibitor is present at a concentration in the range from 0.1 μM to 1 mM. 
     
     
       31. A method as in claim 25, wherein the cells are exposed to the test compound at a concentration from 1 μM to 1 mM. 
     
     
       32. A method as in claim 28, wherein the cells are exposed concurrently to the protease inhibitor and the test compound. 
     
     
       33. A method as in claim 25, wherein the test compound comprises a biological polymer, selected from the group consisting of polypeptides, polynucleotides, and polysaccharides. 
     
     
       34. A method as in claim 25, wherein detecting the change in accumulation comprises: measuring the accumulation of the approximately 22 kD preamyloid intermediate in the cells over a preselected time interval;   measuring the accumulation of the approximately 22 kD preamyloid intermediate in the control cells over the preselected time interval; and   comparing the two measured values.   
     
     
       35. A method as in claim 34, wherein the accumulation of the approximately 22 kD preamyloid intermediate is measured by immunoprecipitation followed by gel electrophoresis. 
     
     
       36. A method as in claim 34, where the preselected time interval is from 0.5 to 24 hours. 
     
     
       37. A method as in claim 34, wherein the exposure to the test compound results in an increase in the measured value of accumulation in the cells exposed to the test compound compared to the cells not exposed to the test compound. 
     
     
       38. A method as in claim 34, wherein exposure to the test compound results in a decrease in the measured value of accumulation in the cells exposed to the test compound compared to the cells not exposed to the test compound.